The phenomenon of homologous recombination of nucleic acids involves physical breaking and crosswise rejoining of nucleic acid strands within homologous sequences. Recombination and gene conversion in mammalian cells have been studied by many groups who have monitored the reconstruction of selectable genes after infection with appropriately constructed viral or plasmid substrates. (Chakrabarti et al., Mol. Cell. Biol. 6:2520-2526, 1986). The results of these experiments indicate that cells efficiently support both intra- and intermolecular recombination and gene conversion. (Id.) Intermolecular recombination refers to recombination between homologous sequences present on two different nucleic acid molecules, while intramolecular recombination refers to recombination between homologous sequences present on a single nucleic acid molecule.
Intermolecular recombination can occur between genes in a plasmid or virus and homologous sequences within a cell. (Miller et al., Mol. Cell. Biol. 6:2895-2902, 1986.) This type of recombination can cause the generation of an infectious virus from an attenuated virus. Fuller et al. codon-optimized the separated sequences of the HIV-1 gag and the HIV-1 pol gene to increase its expression in mammalian cells. These optimizations also reduced identity of nucleotides in an overlapping region of about 200 base pairs present in the gag-pol gene of HIV, which also resulted in reduced levels of intermolecular recombination between the gag and pol open reading frames placed on two independent plasmids and the truncated gag gene contained in a recombinant retroviral vector. (Fuller et al., Hum. Gene Ther. 12:2081-2093, 2001.)
Intramolecular recombination can occur with vectors in which duplicated regions of a gene or a gene fragment are present as direct repeats separated by intervening sequences. This type of recombination generally results in the deletion of the intervening sequences and one copy of the repeated sequences. The frequency of intramolecular recombination is generally a great deal higher than for intermolecular recombination.
The level of intramolecular recombination within a plasmid vector has been quantitated in mammalian cells. (Rubnitz and Subrami, Mol. Cell. Biol. 4:2253-2258, 1984.) Depending upon the size of the homologous regions, the frequency of intramolecular recombination within a transfected plasmid DNA varied between 0.306% and 0.002%. (Id.) Low recombination efficiencies were seen with as little as 14 bases of homology. (Id.)
Intramolecular recombination between homologous sequences has been also documented in a number of animal viruses including picornaviruses, influenza virus, adenovirus, and poxviruses. (Gritz et al., J. Virol. 64:5948-5957, 1990). In vaccinia viruses, it has been shown that tandemly duplicated sequences are genetically unstable. (Id.) In viruses, a level of intramolecular recombination has been seen that is much higher than that seen with plasmid vectors.
For example, in a retrovirus, the frequency of recombination between two identical sequences in the same RNA molecule was found to be about 62%. (Zhang et al., J. Virol. 75:6348-6358, 2001). 99% of these recombinations were intramolecular (between two sequences on one RNA molecule), as opposed to intermolecular (between two RNA molecules). (Id.) With adeno-associated virus, intramolecular recombination was also found to be far more efficient than intermolecular recombination. (Choi et al., J. Virol. 79:6801-6807, 2005). Herpes simplex virus type 1 has also been shown to exhibit high levels of recombination. (Dutch et al., J. Virol. 66:277-285.) In poxviruses, a high frequency of homologous recombination has been seen. An experimental system was used to measure recombination in a vaccinia virus by placing a thymidine kinase (tk) gene between two direct repeats of 1.5 kb of DNA. (Ball, J. Virol. 61:1788-1795, 1987.) During each of the first eight passages under non-selective conditions, 40% of tk+ vaccinia viruses lost their tk+phenotype. (Id.) Under non-selective conditions, the tk− virus increased to an abundance of 99.73% of the total virus population. (Id.) Even under selective conditions, recombination occurred with such high frequency that the majority of infectious virus particles that could be isolate from single plaques contained DNA that had already undergone recombination with subsequent loss of the tk gene. (Id.) Using a recombinant vaccinia virus designed to express three heterologous genes, all expressed from VV p7.5-promoters, Howley et al., Gene 172:233-237, 1996, demonstrated recombination between the repeated promoter sequences. A vaccinia virus recombinant designed to contain a C-repeat region (CRR) from the M protein of Streptococcus pyogenes contained a complex mixture of variants containing from 1 to more than 20 copies of the CRR. (Hruby et al., P.N.A.S. 88:3190-3194, 1991.)
Although it has been shown that multiple genes with homology of about 60-75% inserted into different insertion sites of MVA resulted in a stable multiple recombinant virus (WO 03/097846), there is, however, a need in the art for compositions and methods that reduce the level of intramolecular recombination in vectors, such as, e.g., viral vectors to allow the generation of stable vectors including multiple homologous nucleotide sequences containing longer stretches of identity.